Mar 16, 1988 - increased levels of branched-chain a-keto acids (Schauder,. 1984) and a more reduced mitochondrial NAD pool (Peret et al., 1981; Robinson ...
Vol . 264, No. 6, Issue of February 25, pp. 3347-3351,1989 Printed in U.S. A.
OF BIOLOGICAL CHEMISTRY THEJOURNAL 0 1989 by The American Society for Biochemistry and Molecular Biology, Inc.
Regulation of Hepatic Glycine Catabolism by Glucagon* (Received for publication, March 16, 1988)
Markandeya Jois, Beatrice Hall, Karen Fewer, and John T.BrosnanS From the Department of Biochemistry, Memorial University of Newfoundland, St. John's, Newfoundland, Canada, A l B 3x9
Glucagon stimulates 14C02 production from [1-14C] glycine by isolated rat hepatocytes. Maximal stimulation (70%)of decarboxylation of glycine by hepatocytes wasachieved when theconcentration of glucagon in the medium reached 10 nM; half-maximal stimulation occurred at a concentration of about 2 nM. A lag period of 10 min was observed before the stimulation could be measured.Inclusion of B-hydroxybutyrate(10 mM) or acetoacetate (10 mM) did not affect the magnitude of stimulation suggesting that the effects of glucagon were independent of mitochondrial redox state. Glucagon did not affect either the concentration or specific activity of intracellular glycine, thus excluding the possibilities that altered concentration or specific activityof intracellular glycine contributes to the observed stimulation. The stimulation of decarboxylation of glycine by glucagon was further studied by monitoring 14C02 production from [ l-14C]glycine by mitochondria isolated from rats previously injected with glucagon. Glycine decarboxylation was significantly stimulated in the mitochondriaisolated from the glucagon-injected rats. We suggest that glucagon is a major regulator of hepatic glycine metabolism through the glycine cleavage enzyme system and may be responsible for the increased hepatic glycine removal observed in animals fed high-protein diets.
The catabolism of glycine in mammals occurs primarily in hepatic mitochondria via the glycine cleavage enzyme system (glycine synthase) (EC 2.1.2.10) (Yoshida and Kikuchi, 1970, 1972, 1973). This enzyme systemconsists of fourenzyme proteins: a pyridoxal-containing protein, glycine decarboxylase (Higara and Kikuchi,1980), a lipoic acid-containing protein,aminomethyltransferase(Fujiwara et al., 1979),a 'N,"N-methylene tetrahydrofolate synthesizing protein (Motokawa and Kikuchi, 1974a), and a flavoprotein, dihydrolipoyl dehydrogenase(Motokawa andKikuchi, 197413). The four componentsappeartobepresent as an enzyme complex (Higara et al., 1972) located in the inner mitochondrial compartment (Motokawa and Kikuchi, 1971). The overall reaction catalyzed by this enzyme system is:
mationis available onits regulation.O'Brien(1978), and subsequently Kochi et al. (1986) reported inhibition of the glycine cleavage enzyme system by branched-chain a-keto acids. Recently, Hampson et al. (1983, 1984) have proposed that regulation of glycine cleavage enzyme activity occurs by changesintheoxidation-reductionstate of mitochondrial pyridine nucleotides. Processes which lead to reductionof the mitochondrial NAD(H) and NADP(H) redox couples inhibited metabolic flux through glycine cleavage enzyme. After a high protein meal, the circulating concentrationof glycine is lowered while the concentrations of other amino acids are appreciably elevated (Ishikawa, 1976). The concentration of glycine is decreased in the liver despiteincreaseduptake, suggesting that the increased metabolism of glycine is not secondarytoincreasedplasmamembranetransport.Increased productionof '*C02from [ l-'*C]glycine by liver slices (Matsuda et al., 1973) and isolated hepatocytes (Petzke et al., 1986) has been demonstrated after feeding a high protein diet. These changes cannot be explained by the regulatory mechanisms proposed by O'Brien (1978) or by Hampson et al. (1983, 1984) as feeding a high protein diet is accompanied by increased levels of branched-chain a-keto acids (Schauder, 1984) and a more reduced mitochondrial NAD pool (Peret et al., 1981; Robinson et al., 1981), both of which would be expected toinhibit glycine cleavage activity. Thestudies reported here explore the possibility of hormonal regulation of the glycine cleavage enzyme system. Glucagon has been shown to reduce the intracellular concentration as well as output of glycine in perfused rat liver despite an increased hepatic protein breakdown (Mallette et al., 1969). The studies presented here show that glucagon stimulates flux through the glycine cleavage system. The mechanism of stimulation appears to be independent of mitochondrial redox state and involves a fairly stable alteration in the mitochondrial capacity tocatabolize glycine. EXPERIMENTAL PROCEDURES
M~teriak-[l-'~C]Glycine,[2-"C]glycine, [U-"C]sucrose and scintillation fluids (Aquasol-2 and Omnifluor) were obtained from DuPont-New England Nuclear. Collagenase was obtained from Boehringer Mannheim. Glucagon was obtained from Sigma. Acetoacetate and D-3-hydroxybutyratewere prepared from hydrolysis of ethyl acetoacetate and R-(-)-methyl-3-hydroxybutyrateat room temperature glycine + NAD' + tetrahydrofolate+COz + NH3 + 5N,'0Nfor 3 h by 10% molar excess of sodium hydroxide followed by neutralization with hydrochloric acid and lyophilization. All other remethylene tetrahydrofolate + NADH + H' agents were of analytical grade. Preparation and Incubation of Hepatocytes-Hepatocytes were preAlthough the structure and mechanism of action of the glycine pared as described previously (Krebs et al., 1974) except that hyalucleavage enzyme system have been extensively studied, espe- ronidase was omitted from the perfusate. Cell viability was detercially by Kikuchi andco-workers (Kikuchi, 1973), little infor- mined by 0.2% trypan blue exclusion. Incubations were carried out, in triplicate, in a total volume of 2 ml of Krebs-Henseleit medium, * This work was supported by a grant from the Medical Research gassed with OZ/CO~ (95:5), a t 37 "C in 25-ml Erlenmeyer flasks. For Council of Canada. The costs of publication of thisarticle were determination of decarboxylation rate of glycine, the incubation defrayed in part by the payment of page charges. This article must flasks were equipped with rubber stoppers into which plastic center therefore be hereby marked "aduertisement" in accordance with 18 wells suspended. NCS tissue solubilizer was introduced into center U.S.C. Section 1734 solelyto indicate this fact. wells just before termination of incubation with 0.2 ml of 30% (w/v) $ T o whom correspondence should be addressed. perchloric acid. Glucagon added to the incubation was dissolved in
3347
3348 10 lent
Regulation of Hepatic Glycine Catabolism
hydrochloric acid. Control incubations contained an equivaamount of the acid. Glycogenolysis was measured as glucose release from hepatocytes isolated from fed rats. The perfusion medium contained 20 mM glucose. Gluconeogenesis was measured in hepatocytes isolated from 48-h starved rats, in presence of pyruvate (5 mM) and lactate (5 mM). Preparation and Incubation of Mitochondria-Mitochondria were isolated from male Sprague-Dawley rats (200-250 g) fed a standard pellet diet ad lib&m. The liver was homogenized in a medium (Hampson et al., 1983) containing mannitol, 0.225 M; sucrose, 0.075 M and EGTA,’ 0.1 mM, pH 7.4. The homogenate was centrifuged for 10 min at 2200 X g. Mitochondria were separated from the supernant by centrifugation at 8200 x g for 10 min. The pellet was resuspended in a similar medium without EGTA, containing HEPES, 5.0 mM and pH 7.4. The resuspension and centrifugation steps were repeated three times and the final pellet was resuspended in a small volume of the second medium (without EGTA) to give a concentration of 6080 mg of mitochondrial protein/ml. The respiratory control ratio was determined using a Clark oxygen electrode with 10 mM a-ketoglutarate as substrate and was greater than 4 in all cases. The protein concentration of the mitochondrial suspension was determined using a biuret procedure (Gornall et al. 1949) with bovine serum albumin as standard. Incubations under state three conditions were carried out in triplicate in 25-ml Erlenmeyer flasks at 30°C with constant agitation. The production of “CO, from [1-i4C]glycine by mitochondria was linear up to 20 min. The incubations were routinely carried out for I5 min. The incubation medium (1 ml) consisted of potassium chloride, 100 mM; mannitol, 50 mM; sucrose, 20 mM; potassium phosphate, IO mM; EGTA, 0.1 mM; magnesium chloride, 1 mM; pyridoxal phosphate, 0.175 mM; ADP, 1 mM; HEPES, 25 mM, pH 7.4, and 1 mg of mitochondrial protein. Flux through the glycine cleavage enzyme was monitored by measuring, in triplicate, the production of 14C0, from [l-‘4C]glycine (Hampson et al., 1983). Center wells suspended inside the incubation flasks contained NCS tissue solubilizer to trap ‘*CO2 released after termination of incubation with 0.2 ml of 30% (w/v) perchloric acid. CO, was collected for 60 min. The center wells were then transfered to scintillation vials containing 15 ml of counting fluid (Omnifluor) and counted. Concentrations and Specific Activities of Intracellular and Extracellulur Glycine-Aliquots (0.45 ml) from hepatocytes preparations being incubated with and without glucagon (100 nM) were used to separate hepatocytes from the medium by centrifugal filtration through silicone oil. The hepatocyte suspension was rapidly transferred to a 1.8ml centrifuge tube containing 0.15 ml of sulfosalicylic acid (30%, w/ v) overlaid with a 0.30-ml silicone layer of density 1.022 g/ml and immediately centrifuged for 10 s in an Eppendorf Microcentrifuge at 8000 x g. A portion of the medium above the silcone layer containing extracellular glycine was used to determine the concentration and specific activity of extracellular glycine. The acid layer below the silicone layer, containing deproteinized hepatocytes, was separated, vortexed, and subsequently used to determine the concentration and specific activity of intracellular glycine. For determination of the concentration and specific activities, the pH of the samples was adjusted to 2.2 with lithium hydroxide, and they were then passed through a 0.22 pm filter (Millipore). The concentration of glycine was measured using a Beckman model 121M amino acid analyzer as described by Lee (1974). In separate runs, fractions containing glycine were collected and counted for radioactivity to determine the specific activity of glycine. In these experiments it was necessary to determine and correct for the extracellular water that was carried down to the acid layer by measuring the amount of [U-“Clsucrose, added to hepatocyte suspension, that was carried down to this layer. The [U-i4C]sucrose was added to the hepatocyte suspension just before centrifugation (Fisher and Pogson, 1984). We also used [U-“Clsucrose to determine the intracellular volume of the hepatocytes. In this case the labeled sucrose was in the acid layer, and its dilution, after spinning the hepatocyte suspension through the silicone layer, was a measure of the total water carried down. Subtraction of the extracellular fluid carried down, as determined above, gave the volume of intracellular fluid. These measurements were carried out in triplicate in parallel experiments without [I-i4C]glycine. The average intracellular water content of hepatocytes was 2.22 pl/mg dry weight. mM
i The abbreviations ylenenitrilo)]tetraacetic zineethanesulfonic acid.
used are: EGTA, [ethylenebis(oxyethacid; HEPES, 4-(2-hydroxyethyl)-l-pipera-
RESULTS Incubation of isolated hepatocytes with glucagon (100 nM) resulted in stimulation of decarboxylation of glycine over a wide range of glycine concentrations, ranging from 0.3 to 40 mM (Fig. 1). The percentage stimulation at low concentrations was about 50% in this experiment but this percentage decreased somewhat at higher unphysiological concentrations. All subsequent experiments were carried out at 0.3 mM glycine since this approximates the portal venous glycine concentration (Brosnan et al., 1983). The data in Fig. 2 are from
experiments in which the effects of cysteamine, a known inhibitor of the glycine cleavage enzyme (Yudkoff et al., 1981), on 14C02 production from [1-‘*C]glycine and [2-%]glycine are examined. Hampson et al. (1983) have shown that in the v1
;
”
1.5
1
I
I
10
20
30
40
GLYCINE lmMl FIG. 1. Effect of 100 nM glucagon on ‘%Oz production by rat hepatocytes from various concentrations of [ 1-‘%]glycine. Hepatocytes were incubated for 30 min as described under “Experimental Procedures.” Results are exnressed as mean f S.E. of three separate experiments. *p < 0.05; control versus glucagon, paired t test.
PJgijhi-+~--l 0.0 0.5
1.0 Cysteomlne
1.5
2.0
(mM)
FIG. 2. Effects of cysteamine on 14C02 production from [1-“‘Clglycine (a, 0) and [2-‘%]glycine (W, 0) by hepatocytes in presence (0, n ) and absence (0, 0) of 100 nM glucagon. Preparation and incubation of hepatocytes are described under “Experimental Procedures.” The concentration of glycine was 0.3 mM. Results are expressed as mean -C S.E. of four separate experiments.
Regulation of Hepatic Glycine Catabolism
3349
isolated perfused liver and in isolated liver mitochondria the al., 1985) in hepatocytes. Half-maximal stimulation of phenrate of 14C02production from [l-14C]glycinewas much higher ylalanine hydroxylase by glucagon occurs at 0.7 nM (Fisher than thatfrom [2-14C]glycine,consistent with the proposition and Pogson, 1984). Half-maximal increase of intracellular that theglycine cleavage enzyme is themajor route for hepatic cyclic AMP is observed at 1.5 nM glucagon (Pilkis et ai., 1975). glycine catabolism. The data in Fig. 2 confirm this. In addi- The time course for the stimulation of decarboxylation of tion, Fig. 2 demonstrates that the decarboxylation of [1-“C] glycine by glucagon is shown in Fig. 4. There was a lag period glycine, in the presence or absence of glucagon, is very sensi- of about 10 min before the effect of glucagon could be meastive to cysteamine, an inhibitor of the glycine cleavage en- ured. zyme. Concentrations of cysteamine at which half-maximal To testwhether the effects of glucagon on glycine clearage inhibition of I4CO2production occurred were 0.083 zk 0.004 system could be brought about by cyclic AMP, isolated hepand 0.087 f 0.009 mM in the absence of glucagon and 0.093 atocytes were incubated with dibutyryl cyclic AMP (0.1 mM). k 0.009 and 0.080 zk 0.009 mM in the presence of glucagon Control incubations contained butyric acid (0.1 mM). 14C02 (100 mM) from [l-’4C]glycineand [2-”C]glycine, respectively. production from [l-14C]glycine was stimulated by 50% in The specificity of cysteamine as aninhibitor of glycine cleav- presence of dibutyryl cyclic AMP. Mean f S.E. ( n = 5 ) age system was studied by testing the compound on glycogen- production of 14C02in control and treated hepatocytes were olysis and gluconeogenesis in isolated hepatocytes. Cysteam- 1.64 2 0.05 and 2.48 f 0.20 nmol/mg dry cells/30 min, ine did not affect either the basal rate or thestimulation by respectively. glucagon (0.1-100 nM) of glycogenolysis and of gluconeogenTo testwhether the effects of glucagon weredue to a change esis from pyruvate ( 5 mM) andlactate ( 5 mM) (datanot in either the poolsize or specific activity of intracellular shown). The production of 14C02from [2-14C]glycineprobably glycine, experiments were conducted to determine the effects occurs via the oxidation of the one carbon moiety of 5N,10Nof glucagon on the intracellular concentration and specific methylenetetrahydrofolate as suggested by Hampson et al. activity of glycine after 30 min of incubation (Table I). Glu(1983). Glucagon also stimulated 14C02production from [2- cagon did not affect either the concentration orspecific activ14C]glycineas would be expected from a stimulation of flux ity of intracellular glycine within the period of incubation. through the glycine cleavageenzyme since this would provide The data for samples taken at 15 min of incubation were increased concentrations of 5N,10N-methylenetetrahydrofol- similar (data not shown). This experiment confirms that the ate. Similarly, cysteamine inhibited I4CO2production from effect of glucagon isnot attributableto altered specific activity [2-I4C]glycineas would beexpected from inhibition of glycine of glycine but to a stimulation of glycine metabolism. There cleavage system. was a significant dilution of glycine within the hepatocytes, The dose-response curve for stimulation of decarboxylation possibly due to unlabeled glycine produced by proteolysis, but by glucagon in hepatocytes incubated with 0.3 mM glycine is this dilution was similar in the presence and absence of presented in Fig. 3. A maximal stimulation of 70% was ob- glucagon. Since the intracellular specific activity of glycine served in presence of 10 nM glucagon and half-maximal stim- was about half of that of extracellular glycine, the true rate ulation occurred at a concentrationof 1.9 nM glucagon. Values of glycine decarboxylation is almost double that calculated of 0.2-0.7 nM have been reported as concentrations of gluca- from the specific activity of the externally added glycine. gon needed for half-maximal stimulation of gluconeogenesis (Feliu et al., 1976; Pilkis et al., 1976; Hutson et al., 1976), l I I I I n * activation of glucagon phosphorylase (Blackmore et al., 1982) v1 3.5 et and increase in concentration of intracellular Ca2+ (Sistare
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CONCENTRAT I ON
FIG. 3. Dose-response curve for glucagon on l4CO2production from [l-14C]glycine.The concentration of glycinewas 0.3 mM. All other conditions were as described under Fig. 1. Results are expressed as mean ? S.E. of six separate experiments. * p < 0.05; compared to control values by paired t test.
5
10
15
20
25
T I M E frnin) FIG. 4. Time course of “COZproduction from [ l-’4C]glycine by hepatocytes in the presence and absence of glucagon. Incubations were performed as described under “Experimental Procedures” for different time intervals in the presence of 100 nM glucagon. Other conditions were the same as described under Fig. 1. Results are expressed as mean S.E. of three separate experiments. * p < 0.05; control uersus glucagon, paired t test.
*
Regulation of Hepatic Glycine Catabolism
3350 TABLE I
h
Effects of glucagon on concentrations and specific activities of intracellular and extracellular glycinein hepatocytes Hepatocytes were incubated with 0.3 mM [1-”Clglycine for 30 min in presence and absence of 100 nM glucagon as described under “Experimental Procedures.” Separation of hepatocytes from incubation medium and measurement of concentrations and specific activities of intracellular and extracellular glycine are described under “Experimental Procedures.” Results are expressed as mean f S.E. of three separate experiments.
Intracellular concentration ( w M ) Extracellular concentration ( p M ) Intracellular specific activity (dpm/nmol) Extracellular specific activity (dpm/nmol)
Control
Glucagon (100nM)
2936 +. 170 360 +. 9 66 f 5
3046 ? 335 339 f 13 69 f 8
116 f 2
C
I
I
I
I
119 f 8
TABLEI1 Effects of glucagon on “CO, production from [l-’4C]glycine by hepatocytes in the presence andabsence of 8-hydroxybutyrate or acetoacetate Hepatocytes were incubated with 0.3 mM [1-“Clglycine for 30 min as described under “Experimental Procedures.” Results are expressed as mean f S.E. of six separate experiments. Values are nmol “CO, Droduced/ma dry cells/30 min. Control
Glucagon (100nM)
Addition
No addition 1.57 f 0.20 2.11 2 0.30* 1.65 -C 0.20* 1.20 f 0.08 8-hydroxybutyrate (10 mM) 2.45 k 0.40* Acetoacetate (10 mM) 1.79 f 0.19 * p < 0.05; control versus glucagon, paired t test. ~~
~
~
4
12 16 8 GLYC I NE mM)
20
FIG. 5. Production of 14C02 from [l-’4C]glycine by mitochondria isolated from rats previously treated with glucagon. Glucagon (0.1 mg/300 g body weight; dissolved in 0.5 M KC1/20 mM Tris base) was injected intraperitoneally 25 min before the rats were killed. Mitochondria were incubated for 15 min as are described under “Experimental Procedures”. Results are expressed as mean f S.E. of six separate experiments. * p C 0.05; control versus glucagon, student’s t test.
~
according to the protocol of Lacey et al. (1981). Glycine decarboxylation by mitochondria maintained in state 3 was Mitochondrial NAD’/NADH ratios canbe varied byadding stimulated about 45% by prior glucagon treatment (Fig. 5). P-hydroxybutyrate and/or acetoacetate to the incubation me-Exclusion of pyridoxal phosphate, which was included in the dium. Excess 8-hydroxybutyrate is converted to acetoacetate incubation medium (Hampson et al., 1983),reduced 14C02 by the mitochondrialenzyme P-hydroxybutyrate dehydrogen- production from [l-’4C]glycine (5 mM) by 28 f 6% and 24 f aseresultinginreduction of theNAD(H) redox couple 4% (mean f S.E., n = 4) by mitochondria isolated from rats whereas excess acetoacetate is converted to P-hydroxybutyr- previouslyinjected withsalineand glucagon, respectively. of 4.7 f 0.5 and 6.9 ate resulting in oxidation of NAD(H) couple. A scheme for Glycine decarboxylation exhibited a VmaX f 0.4 nmol/min/mg protein in mitochondria isolated from the regulation of glycine cleavage enzyme by changesin oxidation-reduction statesof mitochondria has been proposed rats treated with vehicle (control) and glucagon, respectively (Hampson et al., 1983, 1984). Hampson et al. (1984) reported ( p < 0.01, control versus glucagon; Student’s t test). The K,,, that glycine decarboxylation by perfused rat liver was inhib- for glycine was similar in both groups (9.7 f 0.9 and 11.3 f ited by 33% by infusion of 10 mM P-hydroxybutyrate whereas 0.9 mM in control and glucagon groups, respectively). Thus a rather stable infusion of 10 mM acetoacetate did notaffect decarboxylation. administration of glucagon to rats brings about of subsequently isolated mitochondria These authorssuggested that thelack of effect of acetoacetate increase in the capacity infusion reflected the alreadyoxidized state of the mitochon- t o catabolize glycine. drial NAD(H) couple. The present studies (Table 11) with DISCUSSION isolated hepatocytes show a 26% inhibition of glycine decarThis is the first report of stimulation of flux through the boxylation ( p < 0.01) by 10 mM P-hydroxybutyrate whereas 10 mM acetoacetate did not affect glycine decarboxylation. glycine cleavage enzyme system by glucagon. The effects of Glucagon stimulated glycine decarboxylation by 40-50% ir- glucagon appeared tobe stable as evidenced by persistence of respective of the presence orabsence of P-hydroxybutyrate or these effects in mitochondria isolated from rats treated with glucagon even though the mitochondria were washed three acetoacetate in the incubationmedium. The experiments reported above do not establish the mech- times during isolation. Two separate mechanisms have been proposed for regulaanism by which glucagon stimulates flux through the glycine cleavage enzyme. Two principal typesof metabolic actions of tion of the activity of the glycine cleavage system. One proglucagon have been described. The classiceffects involve posal suggests inhibition of glycine decarboxylation by covalent modification, byphosphorylation, of key cytoplasmic branched-chain a-keto acids by transfer of reducing equivaenzymes (Garrison et al., 1984). Another set of effects, which lents to theglycine cleavage system through lipoamide dehyare evident in isolated intact mitochondria, do not appear to drogenase, an enzyme protein shared by the glycine cleavage acid dehydrogenase involve phosphorylation (Siesset al., 1979; Vargas etal., 1982). system and the branched-chain a-keto We therefore carried out a set of experiments to determine complex (O’Brien, 1978and Kochi et al., 1986). O’Brien (1978) and Kochi et al. (1986) incubated mitochondria with whether the glycine cleavage system would be activated in mitochondria isolated from rats injected with glucagon (0.1 branched-chain a-ketoacid at concentrations of 2 and 5 mM, mg/300 g body weight, intraperitoneal) 25 min previously respectively, and showed inhibition of glycine cleavage system
Reguhtion of Hepatic Glycine Catabolism 40-50%. by However physiological concentrations of branched-chain a-keto acids arein the micromolar range (Schauder, 1984). In view of reports of stimulation of branched-chain a-keto acid dehydrogenase activity and of an increase in the circulating concentration of branched-chain a-keto acids by exogenous glucagon (Schauder, 1984), the glycinecleavage system activity wouldbe predicted to be inhibited by glucagonin contrast to findings the of the present study. It is probable that thebranched-chain a-keto acids do not inhibit the glycine cleavage system under physiological conditions. The second mechanism, proposed by Hampson et al. (1983, 1984), involves regulation of the glycine cleavage system by changes in oxidation-reduction states of mitochondrial pyridine nucleotide. Theseauthors showed high sensitivity of glycine decarboxylation activity to uncouplers and reducing substrates. In general, substrates and agents that reduce intramitochondrial pyridine nucleotides inhibit flux through the glycine cleavage enzyme system. However, stimulation of the glycine cleavage system by glucagon cannot be explained by this mechanism because glucagon results in reduced redox states of both the cytosolic and mitochondrial pyridine nucleotides (Williamson et al., 1969; Sugano et al., 1980; Kimura et al., 1984; Balaban and Blum, 1982). That the effects of glucagon on the glycinecleavage system areindependent of mitochondrial redox state is also evident from the fact that glucagon equally stimulated the glycine cleavage system in the presence or absence of b-hydroxybutyrate or acetoacetate. It is also of interest to note that after feeding a high protein diet, the hepatic glycine decarboxylation rate increases (Matsuda et al., 1973; Petzke et al., 1986) even though the mitochondrial NAD/NADH ratio decreases (Peret et al., 1981; Robinson et al., 1981). It is possible that the changes seen in glycine metabolism after feeding high protein diets are mediated by glucagon. Feeding high protein diets results in an increased release of glucagon and an increased glucagon/ insulin ratio (Peret et a!., 1981). There seem to be two distinct mechanisms of action of glucagon on hepatic metabolism. One is the covalent modification, by phosphorylation, of cytoplasmic enzymes. This occurs by activation of the cyclic AMP-stimulated protein kinase and target enzymes include glycogen phosphorylase, pyruvate kinase, and phenylalanine hydroxylase (Garrison et al., 1984). These effects are typically labile in that they are readily reversible (via phosphatases) upon removal of the hormone. The second type of glucagon actionstimulates mitochondrial metabolism of a variety of substrates and appears to. be fairly stable in that the effects persist for some time after the removal of the hormone and, indeed, remain evident in mitochondria that have been isolated and washed without any precautions taken to preserve the phosphorylation states of proteins (see Halestrap, 1986). Typically these phenomena are evident only in intact mitochondria and include such effects as increased glutaminase activity, increased citrulline synthesis, and increased pyruvate transport. These effects can be brought about by cyclicAMP although the link between the intramitochondrial functions and increased cytoplasmic CAMP has not been established. The stimulation of glycine metabolism byglucagon clearly belongs to this
3351
second type of action. The effect is rather long lived in that it persists in mitochondria isolated 'from glucagon injected animals. The mechanism of action of glucagon on theglycine cleavage enzyme system cannot be identified from these studies. Nevertheless, the present observations have considerable physiological relevance and may explain the very rapid hepatic metabolism of glycine that is known to occur in animals ingesting high protein diets. Acknowledgment-We acid analysis.
thank D.E. Hall for carrying out amino
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